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CARDIOVASCULAR
Department of Pharmacology and Toxicology, Medical College of Wisconsin, Milwaukee, Wisconsin (W.Y., C.J.H., W.B.C.); and Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas, Texas (V.R.T., S.A., J.R.F.)
Received for publication
August 1, 2007
Accepted
December 21, 2007.
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Abstract |
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 Top Abstract Materials and MethodsResultsDiscussionReferences |
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14,15-EET » 15-hydroxyeicosatetraenoic acid > 14,15-EET-thiirane >14,15-dihydroxyeicosatrienoic acid. This order is in agreement with the efficacy and potency of cAMP production. In summary, 20-125I-14,15-EE8ZE is a radiolabeled EET agonist that is useful to study binding and metabolism. Using this radioligand, we have identified a specific high-affinity and high-abundance EET binding site in U937 cell membranes. This binding site could represent a specific EET receptor, which is probably a G protein-coupled receptor.
; Fisslthaler et al., 1999; Campbell and Falck, 2007). They regulate vascular tone in a paracrine manner. The vascular endothelium releases EETs upon stimulation with bradykinin, acetylcholine, arachidonic acid, flow, or cyclic stretch (Campbell and Falck, 2007). EETs activate smooth muscle membrane large conductance, calcium-activated potassium (BKCa) channels to cause hyperpolarization and vascular relaxation (Campbell et al., 1996; Fisslthaler et al., 1999; Campbell and Falck, 2007). EETs are also key regulators of vascular homeostasis. They inhibit cytokine-mediated nuclear factor-
B activation, which results in inhibition of endothelial adhesion molecule expression and monocyte adhesion to vascular endothelium (Node et al., 1999). However, the initial step of EET signaling is unknown.
There are specific structural requirements of the EET molecules for bovine coronary artery relaxation (Falck et al., 2003a) and inhibition of vascular cell adhesion molecule-1 expression (Falck et al., 2003b). For vasorelaxation, the minimal structure for full agonist activity is 14(S), 15(R)-cis-epoxy-eicosa-8Z-enoic acid (14,15-EE8ZE). Changing the position of double bonds in the EET molecule can result in antagonist activity (Gauthier et al., 2002). For example, 14,15-epoxyeicosa-5Z-enoic acid (14,15-EE5ZE) is an EET antagonist. Such structure-activity relationships indicate that a binding interaction is involved in the mechanism of action that requires precise conformation of the EET molecules. Several studies have suggested that EETs act at a plasma membrane receptor or binding site. 11,12-EET activates coronary smooth muscle BKCa channels in inside-out patches where a small portion of plasma membrane was excised (Li and Campbell, 1997). In aortic smooth muscle cells, a membrane-impermeable 14,15-EET derivative was equipotent to 14,15-EET in inhibiting aromatase activity (Snyder et al., 2002). Radioligand binding studies using [3H]14,15-EET revealed high-affinity, saturable specific binding in intact monocytes, U937 cells, and mononuclear cell membrane fractions (Wong et al., 1993, 1997, 2000).
Guanine nucleotide-binding (G) protein, Gs, activation plays a key role in EET actions. In inside-out patches, EET-mediated smooth muscle BKCa activation requires intracellular GTP, but not ATP. EET-mediated BKCa activation can be blocked by the G protein inhibitor guanosine 5'-O-(2-thio)diphosphate and an antibody against Gs
, but not antibodies against Gi or Gβ
(Li and Campbell, 1997). EETs promote GTP binding to Gs but not Gi in endothelial cells (Node et al., 2001). Because coronary smooth muscle BKCa channels can be activated directly by GTP-activated Gs (Scornik et al., 1993), it is possible that EETs activate BKCa through a membrane-delimited action of Gs. In addition, EETs activate a classic Gs signaling cascade. They activate adenylyl cyclase, increase intracellular cAMP levels, activate protein kinase A and the cAMP response element-binding protein (Node et al., 2001; Carroll et al., 2006; Spector and Norris, 2007). Because heterotrimeric G proteins characteristically couple to a membrane receptor, these data suggest that EETs initiate their signaling cascades through a membrane, Gs-coupled receptor. However, such a receptor has not been identified at a molecular level.
Characterization of an EET receptor/binding site has been hindered by the active metabolism of EETs. EETs are rapidly esterified into phospholipids via the action of fatty acyl CoA synthase and acyltransferase and hydrolyzed to the corresponding vicinal-dihydroxyeicosatrienoic acids (DHETs) by soluble epoxide hydrolase (sEH) (Spector and Norris, 2007). They are also converted to shorter chain fatty acids by β-oxidation (Spector and Norris, 2007). The lack of commercially manufactured radiolabeled EETs has also limited radioligand binding studies of an EET receptor. Currently, 14C- or 3H-labeled EETs may be synthesized from radiolabeled arachidonic acid. This synthesis is inefficient and costly. The yields are less than 50% due to the production of varying amounts of all four EET regioisomers and the formation of diepoxides (Corey et al., 1979). Thus, there is a need for an EET analog that can be radiolabeled efficiently, inexpensively, and with high specific activity. In the present study, we developed the radioiodinated EET agonist 20-125I-14,15-EE8ZE as a radioligand to characterize EET binding to cell membranes. Membranes from U937 cells were used to characterize the ligand binding site since previous studies showed that [3H]14,15-EET binds U937 cells with high affinity (Wong et al., 1997) and to represent a defined system for testing radioligand binding with EET analogs.
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Materials and Methods |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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; Cai et al., 2006). 20-125I-14,15-EE8ZE is synthesized from its corresponding 20-OTs-14,15-EE8ZE as reported previously (Prestwich et al., 1988). To a pH 9 to 10 solution of carrier-free Na125I (0.8 nmol/20 µl; 2 mCi; 17.4 Ci/mg) was added NaI solution (6.4 µg/20 µl in acetone) followed by 20-OTs-14,15-EE8ZE (640 nmol/40 µl in acetone). The lead-shield reaction vial was warmed to 40°C, and then it was shaken twice daily for 5 days. The reaction mixture was put directly onto a Bio-Sil A (Bio-Rad, Hercules, CA) silicic acid column. The column was then eluted by 2 volumes of 10% ethyl acetate in hexane and 2 volumes of 20% ethyl acetate in hexane. The flow-through was collected, dried under N2, and purified by reverse-phase-high performance liquid chromatography (HPLC). The residues were redissolved in 100 µl acetonitrile/glacial acetic acid (999:1) and 100 µl of distilled H2O, and then they were resolved on a Nucleosil C18 reverse-phase column (5 µm; 4.6 x 250 mm; Phenomenex, Torrance, CA). Solvent A was distilled H2O and solvent B was acetonitrile/glacial acetic acid (999:1). A linear gradient from 70% solvent B in solvent A to 73% solvent B over 40 min was used at a flow rate of 1 ml/min. The column effluent corresponding to the elution time of synthetic 20-I-14,15-EE8ZE was collected, extracted, and dried under N2. The specific activity of 20-125I-14,15-EE8ZE was 47.69 Ci/mmol. Culture of U937 Cells and Membrane Preparation. U937 cells were cultured in suspension in RPMI 1640 medium (Invitrogen, Carlsbad, CA) medium supplemented with 10% fetal bovine serum (HyClone Laboratories, Logan, UT), 25 mM HEPES, 2 mM L-glutamine, and 1 mM sodium pyruvate, 100 U/ml penicillin, 100 µg/ml streptomycin, and 0.25 µg/ml amphotericin B. The cultures were maintained at a density of 5 to 10 x 105 cells/ml at 37°C in a humidified atmosphere containing 5% CO2. To prepare the membrane fraction, cells were collected by centrifugation at 1000 rpm for 2 min and washed twice with Hanks' balanced salt solution without Ca2+ or Mg2+. The pellet was resuspended with Hanks' balanced salt solution containing protease inhibitor cocktail (Roche Diagnostics, Indianapolis, IN), and then it was sonicated for 20-s bursts on ice five times at setting 4. The homogenate was centrifuged at 1000g for 10 min at 4°C to remove unbroken cells. The supernatant was centrifuged at 150,000g for 45 min at 4°C. The pellet represents the membrane fraction and was resuspended in 50 mM HEPES, pH 7.4.
Western Immunoblotting. To determine the composition of the membrane fraction, the membrane fraction proteins (25 µg) were electrophoretically separated on 12% polyacrylamide Redi-Gels (Bio-Rad) at 100 V for 90 min. Proteins were transferred to nitrocellulose membranes (Bio-Rad), and the membranes were incubated in Tris-buffered saline containing 0.1% Tween 20 and 5% milk (TBSTM) at room temperature for 3 h. After washing, the membranes were incubated overnight at 4°C with a primary antibody in TBSTM solution. Membranes were washed and incubated at room temperature for 1 h with an IgG-horseradish peroxidase-conjugated secondary antibody (1:2000) in TBSTM solution. Immunoreactive bands were detected by a chemiluminescence detection kit (Pierce Chemical, Rockford, IL) and photographic film (BioMax; Eastman Kodak, Rochester, NY). The following primary antibodies and dilutions were used to characterize the membrane fraction: cytochrome P450 reductase, 1:1000 (Abcam Inc., Cambridge, MA); nucleoporin, 1:500 (Santa Cruz Biotechnology, Inc.); ATP synthase β, 0.2 µg/ml (Invitrogen); ATP synthase complex 3, core 1, 0.1 µg/ml (Invitrogen); ATP synthase complex 3, core 2, 0.4 µg/ml (Invitrogen); caveolin 1, 2 µg/ml (Millipore, Billerica, MA); pan cadherin, 1:2500 (Abcam Inc.); and Na+K+ ATPase, 1:5000 (Abcam Inc.).
cAMP Assay. U937 cells were resuspended in RPMI 1640 medium with 25 mM HEPES, pH 7.4, 20 µM Ro 20,1724, a phosphodiesterase inhibitor, and 1 µM adamantyl dodecanoic acid urea (AUDA), an sEH inhibitor. Cells were pretreated with 20 µM triacsin C, an acyl CoA transferase inhibitor, for 30 min and with 10 µM miconazole, a P450 inhibitor, for 10 min at 37°C. The cells were transferred into polypropylene tubes at a density of 106 cells/ml. Ligands or vehicle was incubated with cells (250,000 cells/incubation) for 10 min at 37°C. For antagonist studies, cells were pretreated with 10 µM 14,15-EE5ZE or equal amount of ethanol for 10 min at 37°C before adding 11,12-EET or 20-I-14,15-EE8ZE. The assays were terminated by adding 500 µl of ice-cold 0.15 N HCl. The amount of cAMP in the acid extract was determined by radioimmunoassay according to the manufacturer's instructions (GE Healthcare, Chalfont St. Giles, UK). Neither inhibitors nor ligands interfered with the assay. The data are expressed as -fold increase compared with vehicle control. The EC50 and Emax values were determined by nonlinear regression to fit the data to a sigmoidal concentration-response equation using Prism software (GraphPad Software Inc., San Diego, CA). Statistical evaluation of the data was performed by t test. P < 0.05 was considered statistically significant.
Vascular Reactivity of Bovine Coronary Arteries. Bovine hearts were purchased from a local slaughterhouse. The left anterior descending coronary artery was dissected and cleaned of connective tissue. Arteries of 2-mm diameter were cut into rings (3 mm in width), and they were suspended on a pair of stainless hooks in a 6-ml water-jacketed organ chamber in Krebs' buffer consisting of 119 mM NaCl, 4.8 mM KCl, 24 mM NaHCO3, 0.2 mM KH2PO4, 0.2 mM MgSO4, 11 mM glucose, 0.02 mM EDTA, and 3.2 mM CaCl2. The buffer was equilibrated with 95% O2, 5% CO2, and they were maintained at 37°C. Tensions were recorded as described previously (Campbell et al., 1996). In brief, submaximal concentrations of a thromboxane-mimetic U46619
[GenBank]
(10–20 nM) were administered to contract the vessels to 50 to 75% of KCl-induced contraction. Increasing concentrations of 14,15-EET or 20-I-14,15-EE8ZE were added to the chamber. In BKCa blockade studies, the arteries were incubated with 100 nM iberiotoxin (IBTX) for 10 min before U46619
[GenBank]
contraction. In the high extracellular potassium ([K+]o) studies, K+ was increased to 20 mM by substitution of Na+. Results are expressed as percentage of relaxation (means ± S.E.M.), with 100% relaxation representing basal tension. Statistical evaluation of the data was performed by a one-way analysis of variance followed by the Student-Newman-Keuls multiple comparison test if justified by the analysis of variance results. P < 0.05 was considered statistically significant.
Metabolism of 20-125I-14,15-EE8ZE. 20-125I-14,15-EE8ZE (24 nM) was incubated with 50 µg of U937 cell membrane protein for 15 min at 4°C in 200 µl of binding buffer consisting of 20 mM HEPES, 10 mM CaCl2, and 10 mM MgCl2, at pH 7.4. The metabolites were harvested by liquid-liquid extraction using 800 chloroform/methanol (1:2) followed by addition of 268 µl of chloroform and 240 µl of H2O. Radioactivity of the organic and aqueous phases was measured in a Packard Cobra-II auto-gamma counter. A 200-µl aliquot of the organic fraction was collected and dried under N2. The extracts were then analyzed by reverse-phase HPLC as described above, except that the linear gradient was from 50% solvent B in solvent A to 94% solvent B within 40 min. Column effluent was collected in 0.5-ml fractions, and the radioactivity was measured.
20-125I-14,15-EE8ZE Binding Assays. 20-125I-14,15-EE8ZE binding assays were carried out using a 48-well harvester system (Brandel Inc., Gaithersburg, MD). U937 membranes (50 µg protein/incubation) were incubated with 20-125I-14,15-EE8ZE in binding buffer in the presence of 20 µM 14,15-EEZE (nonspecific binding) or an equivalent amount of ethanol (total binding) at 4°C with shaking. Incubations (total volume 200 µl) were terminated by filtration through GF/B glass filter paper (Whatman, Clifton, NJ), followed by five washes with 5 ml of ice-cold binding buffer. Radioactivity remaining on the filters was measured by a gamma-counter. Specific binding was defined as total binding minus nonspecific binding.
For saturation studies, 20-125I-14,15-EE8ZE (1–55 nM) was incubated with U937 membrane protein (50 µg) for 15 min. For the time course study, 23 nM 20-125I-14,15-EE8ZE was incubated for various times (1–30 min). For the reversibility study, 16 nM 20-125I-14,15-EE8ZE was incubated for 10 min, after which 1 or 20 µM unlabeled 11,12-EET was added, and the incubation was continued for various times (7–60 s). For the competition studies, 20-125I-14,15-EE8ZE (11–19 nM) was incubated in the presence of increasing concentrations of unlabeled competing ligands (0.1 nM–100 µM) or vehicle (ethanol). Specific binding obtained in the presence of vehicle was defined as 100%. For the GTPS study, saturation isotherms were determined in the presence or absence of 0.5 or 10 µM GTPS.
The data were analyzed using Prism software. The rate of association was determined from time course studies using nonlinear regression to fit the data to the one phase exponential association equation. The equilibrium dissociation constant (KD) and maximal binding site density (Bmax) values were determined from saturation studies using nonlinear regression to fit the data to the single-site, equilibrium binding equation. The IC50 values of competing ligands were calculated using nonlinear regression to fit the data to a one-site competition equation. The Ki values were calculated from the IC50 values using the equation of Cheng and Prusoff (1973).
Chemicals. Carrier-free Na125I was obtained from GE Healthcare. U46619 [GenBank] was obtained from Cayman Chemical (Ann Arbor, MI). Iberiotoxin, triacsin C, and miconazole were obtained from Sigma-Aldrich (St. Louis, MO). Ro 20,1724 was obtained from BIOMOL Research Laboratories (Plymouth Meeting, PA).
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Results |
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5 and 11 double bonds from 14,15-EET does not alter vascular relaxation and that 14,15-EE8ZE (structure in Fig. 1) mimics the relaxation effects of 14,15-EET in bovine coronary artery rings (Falck et al., 2003a). Based on this knowledge, 20-I-14,15-EE8ZE, an iodinated analog of 14,15-EE8ZE, was developed (structure in Fig. 1). This compound differs from 14,15-EE8ZE in that an iodide is substituted for a hydrogen atom in the 20-methyl group.
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) could inhibit the EET-induced cAMP production. 11,12-EET-mediated cAMP accumulation was significantly inhibited by 10 µM 14,15-EE5ZE (Fig. 2B). Likewise, 10 µM 14,15-EE5ZE inhibited the cAMP accumulation produced by 30 µM 20-I-14,15-EE8ZE (Fig. 2C). These data indicate that 20-I-14,15-EE8ZE activates a receptor/signaling pathway similar to 11,12-EET in U937 cells, although it has lower efficacy. In contrast, neither 14,15-DHET, the sEH metabolite of 14,15-EET, nor 15-HETE, the 15-lipoxygenase metabolite of arachidonic acid, increased cAMP production in U937 cells (Fig. 2A).
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). The EC50 values are approximately 1 µM, and the maximum relaxations average 80 to 94% at 10 µM. Likewise, 20-I-14,15-EE8ZE relaxed U46619
[GenBank]
-precontracted arteries, with maximum relaxation of 84.7 ± 1.7% and an EC50 value of 1 µM (Fig. 3A). 14,15-EET and 11,12-EET activate BKCa channels, leading to hyper-polarization and relaxation of the smooth muscle. To examine the role of K+ channel inhibition in the 20-I-14,15-EE8ZE-mediated relaxations, extracellular K+ was increased from 4 to 20 mM. 20-I-14,15-EE8ZE relaxation was significantly reduced (maximum relaxation = 8.3 ± 1.6%; Fig. 3B) in the presence of 20 mM. Likewise, pretreatment with the BKCa channel blocker IBTX at 100 nM eliminated 20-I-14,15-EE8ZE-induced relaxations (Fig. 3B). These results demonstrate that 20-I-14,15-EE8ZE retains full vascular activity compared with the EETs and that it relaxes bovine coronary arteries through similar mechanisms.
Metabolic Stability of 20-125I-14,15-EE8ZE. EETs are metabolized by multiple enzymatic pathways, including epoxide hydration, phospholipid esterification and β-oxidation (Spector and Norris, 2007). Likewise, 20-I-14,15-EE8ZE is susceptible to metabolism because it contains a C1 carboxyl and a 14,15-epoxide group. We investigated the stability of 20-125I-14,15-EE8ZE under the experimental conditions of radioligand binding. After a 15-min incubation with U937 membranes at 4°C, the majority of radioactivity (92.9 ± 0.2%) was extracted with the organic solvent. Incubation of 20-125I-14,15-EE8ZE with either boiled or control U937 membrane proteins resulted in greater than 92% of the radioactivity remaining in the organic phases (Fig. 4A). HPLC chromatograms of the organic phases showed one major radioactive peak at 27.5 min, which comigrated with the synthetic 20-I-14,15-EE8ZE standard (Fig. 4, B–D). Similar results were achieved in 30-min incubations (data not shown). These results suggest that 20-125I-14,15-EE8ZE is metabolically and chemically stable under the binding conditions.
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Characterization of Membrane Fraction. The protein components of the membrane fraction were characterized by Western immunoblotting with specific antibodies. Five of the Western blots are shown in Fig. 4E; the others are not shown. Immunoreactive bands were detected for plasma membrane markers (Na+/K+ ATPase, 112 kDa; pan cadherin, 125 kDa; and caveolin-1, 22 kDa), an endoplasmic reticulum marker (cytochrome P450 reductase, 78 kDa), mitochondrial membrane markers (ATP synthase complex III core1, 52 kDa; ATP synthase complex III core 2, 48 kDa; and ATP synthase β, 57 kDa), and a nuclear membrane marker (nucleoporin, 63 kDa). Thus, the membrane preparation has a mixture of cellular membranes including plasma membranes.
Characterization of 20-125I-14,15-EE8ZE Binding. Binding of 20-125I-14,15-EE8ZE to U937 membrane proteins was rapid at 4°C. The half-time of association was 2.1 min at 23 nM 20-125I-14,15-EE8ZE. The specific binding reached equilibrium within 10 min, and it remained unchanged up to 30 min (Fig. 5A). Equilibrium binding was performed by incubating increasing concentrations of 20-125I-14,15-EE8ZE with 50 µg of U937 membrane proteins at 4°C for 15 min. Both total and nonspecific binding increased as the radioligand concentration increased (Fig. 5B). Nonspecific binding was relatively high, accounting for 50 to 80% of total binding, and was linearly related to radioligand concentrations. Specific binding was saturable and fit a one-site binding model best, with KD and Bmax of 11.8 ± 1.1 nM and 5.8 ± 0.2 pmol/mg protein, respectively (Fig. 5, C and D).
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The competition for 20-125I-14,15-EE8ZE binding by a series of analogs was determined. The competing analogs include 11,12- and 14,15-EET; 14,15-EE5ZE, an EET antagonist; 14,15-DHET, the sEH metabolite of 14,15-EET; 14,15-EET-thiirane, an inactive 14,15-EET analog; and 15-HETE, the 15-lipoxygenase metabolite of arachidonic acid (for structures, see Fig. 1). All of the resulting isotherms were fit to a one-site competition model and dissociation constants (Ki) were calculated from IC50 values (Fig. 7; Table 1). Both EET regioisomers and 14,15-EEZE produced concentration-dependent inhibition of 20-125I-14,15-EE8ZE binding to U937 membranes. Inhibition potencies were 11,12-EET >14,15-EE5ZE 14,15-EET. 11,12-EET was approximately 3 times more potent than 14,15-EET, with Ki values of 12 and 40 nM, respectively. 14,15-EE5ZE was similar to 14,15-EET, with a Ki of 37 nM. 14,15-DHET, 14,15-EET-thiirane, and 15-HETE were poor competitors of 20-125I-14,15-EE8ZE binding with Ki values of 8800, 2500, and 2400 nM, respectively (Fig. 7; Table 1). The affinities of these analogs to compete with 20-125I-14,15-EE8ZE for binding to U937 membranes correlate, with their efficacies in cAMP activation. These data suggest that EET-induced cAMP production of U937 cells is mediated through binding to a membrane receptor.
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Radioligand binding of agonists to G protein-coupled receptors (GPCR) is usually modulated by uncoupling of G proteins from the receptor. In the presence of 0.5 µM GTPS, the Bmax value of 20-125 I-14,15-EE8ZE binding was significantly decreased, whereas the KD was slightly decreased, but the change was not statistically significant (Fig. 8; Table 2). In the presence of 10 µM GTPS, the specific binding was abolished (Fig. 8). These results suggest that the 20-125I-14,15-EE8ZE binding site is a GPCR.
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Discussion |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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; Carroll et al., 2006; Spector and Norris, 2007). In the current study, EETs increased cAMP accumulation in U937 monocytes in a concentration-dependent manner. 11,12- and 14,15-EET had similar efficacies, but 11,12-EET was 3 times more potent than 14,15-EET. The 11,12-EET-mediated cAMP production was blocked by the EET antagonist 14,15-EE5ZE. Because cAMP is a characteristic second messenger of GPCR-Gs-adenylyl cyclase signaling cascade, these data strongly suggest that EETs bind and activate a membrane Gs-coupled receptor in U937 cells.
Radioligand binding studies of EET receptors have been limited by lack of radiolabeled ligands. Based on the structure of the 14,15-EET mimetic, 14,15-EE8ZE, we developed an iodinated EET agonist, 20-I-14,15-EE8ZE, by substituting a hydrogen at C20 with iodide. The agonist activity of 20-I-14,15-EE8ZE was documented. 20-I-14,15-EEZE stimulated U937 cell cAMP accumulation with similar potency as 11,12-EET, which was blocked by 14,15-EE5ZE. However, the efficacy of 20-I-14,15-EE8ZE was less than 11,12- and 14,15-EET. In bovine coronary arteries, where EETs represent endothelium-derived hyperpolarizing factors and cause vascular relaxation through BKCa channel activation (Campbell et al., 1996), 20-I-14,15-EE8ZE relaxed bovine coronary arterial rings with similar potency and efficacy as the EETs. This activity was inhibited by K+ efflux blockade and a specific BKCa blocker. These results suggest that 20-I-14,15-EE8ZE acts through similar signaling mechanisms as the EETs.
EETs are actively metabolized through epoxide hydration, phospholipid esterification, and β oxidation (Spector and Norris, 2007). These pathways require intracellular enzyme(s) as well as ATP and CoA. In the current radioligand binding studies, ligands were incubated with U937 membrane fractions at 4°C. Because intracellular components are unavailable, the metabolism of EET analogs should be minimal. Indeed, 20-125I-14,15-EE8ZE was metabolically stable under the binding conditions. No hydration or β-oxidation products were observed.
Using 20-125I-14,15-EE8ZE, we characterized EET binding to U937 cell membrane fractions. This membrane fraction contained a mixture of cellular membranes, including plasma membranes. Specific binding was time-dependent and monophasic. The binding site/receptor was in high abundance, with a Bmax of 5.8 ± 0.2 pmol/mg protein. The KD was 11.8 ± 1.1 nM, which is similar to the KD characterized by Wong et al. (1997) of 13.84 ± 2.58 nM using [3H]14,15-EET binding in whole U937 cells. The low nanomolar range of KD value is in agreement with the affinities of many native eicosanoids to their receptors such as prostaglandin E2 to EP1 (Sharif and Davis, 2002) and EP2 (Kiriyama et al., 1997) receptors; prostaglandin I2 to IP receptor (Pimpinelli et al., 1999); and leukotriene C4 to its receptor (Levinson, 1984). Based on the measured KD of 11.8 nM, the dissociation should be very rapid. If we assume an association rate constant (kon) of 0.5 x 106 M–1 ·s–1, the dissociation rate constant (koff = KD · kon) is calculated as 0.06 s–1 and the t1/2 (ln 2/koff) is calculated as 11.6 s. Indeed, specific binding was reversible, and the dissociation was very rapid. With 1000 times excess of 11,12-EET, the specific binding was completely displaced within 1 min. Thus, the 20-125I-14,15-EE8ZE binding site is reversible and of high affinity and high abundance in U937 membranes. The cellular location of the binding site cannot be stated with certainty due to the composition of the membranes studied.
20-125I-14,15-EE8ZE binding was selectively inhibited by EET agonists and a physiological EET antagonist, but not by the inactive EET analogs 14,15-DHET and 14,15-EET-thiirane, or unrelated eicosanoid, 15-HETE. Binding affinities of the competing analogs were consistent with their efficacies in cAMP stimulation. 11,12-EET was 3 times more potent than 14,15-EET in cAMP activation, and the Ki value of 11,12-EET was 3 times lower than 14,15-EET. 14,15-DHET, 14,15-EET-thiirane, and 15-HETE were very poor competitors, with Ki values in the micromolar range, and they did not increase cAMP production. These data indicated that the binding site labeled by 20-125I-14,15-EE8ZE is specific for EETs.
Guanine nucleotides are known to modulate G protein-coupled receptors. With Gs-coupled receptors, G protein uncoupling by unhydrolyzable GTP or constant activation of Gs results in inhibition of high-affinity binding. However, the precise effects on KD and Bmax vary with the G protein coupling mechanism and with the type of receptor, tissue/cell, and species. In rat striatum and P12 cell membranes, agonist binding affinity and capacity of A2A adenosine receptors were inhibited by cholera toxin and 5-guanylylimidodiphosphate (Hide et al., 1992; Mazzoni et al., 1993; Cunha et al., 1999). Alternatively, this modulation was not observed in rabbit (Nanoff et al., 1991) or bovine (Barrington et al., 1989) striatal membranes. β-Adrenergic receptors contain two binding sites with discrete affinities as addressed by agonist competition binding studies. Pretreatment with 5-guanylylimidodiphosphate completely eliminated the high-affinity Gs-coupled binding site of rat and rabbit cardiac (Gando et al., 1997; Makino et al., 2003) and rat neuronal (Morin et al., 1997) β-adrenergic receptors. In the present study, 20-125I-14,15-EE8ZE binding to U937 membranes was reduced by a low concentration (0.5 µM) of GTPS. The Bmax was reduced by 45%, and the KD value was not significantly changed. When the U937 membranes were pretreated with 10 µM GTPS, the 20-125I-14,15-EE8ZE specific binding was abolished. The precise G protein coupling mechanism of the EET receptor remains unclear. Nonetheless, the inhibitory effect of GTPS indicates that the 20-125I-14,15-EEZE binding site is a GPCR.
The biological consequences of EET-mediated elevation of intracellular cAMP in U937 cells were not investigated. However, many cAMP-elevating reagents, including adenosine, forskolin, and phosphodiesterase inhibitors (Eigler et al., 1998), exert anti-inflammatory effects through regulation of cytokine production by monocytes and macrophages. These compounds up-regulate the anti-inflammatory cytokine interleukin-10 and they suppress the proinflammatory cytokine tumor necrosis factor-. It is reasonable to speculate that EETs similarly regulate cytokine balance through activation of monocyte cAMP production.
The Ki values of EETs were in the lower nanomolar range, whereas the concentrations required for cAMP stimulation were micromolar. This low efficacy is surprising in light of previous data showing that nanomolar concentrations of EETs activate cAMP production in endothelial cells (Node et al., 2001) and smooth muscle BKCa channels (Campbell et al., 1996; Li and Campbell, 1997). It is interesting that EET receptors are highly expressed in the monocyte cell line but that they show a low signaling efficiency, which suggests a poor Gs coupling to the EET receptor in U937 cells. This may represent a mechanism regulated by EET bioavailability. Physiological plasma concentration of EETs is approximately 30 nM (Karara et al., 1992). Our data would predict that the EET-Gs-cAMP pathway would be silent under physiological conditions. During inflammation, local blood flow is increased. Increased shear stress (Huang et al., 2005), cyclic stretch (Fisslthaler et al., 2001), and laminar flow (Liu et al., 2005) stimulate endothelial production of EETs. Therefore, EET concentrations could increase locally, triggering monocyte Gs-cAMP pathway activation and thereby regulate cytokine production to prevent tissue injury. In addition, the U937 is a cell line with a leukemia origin. It is possible that the EET receptor G protein coupling is altered during oncogenesis. Future investigations with normal cell lines will shed light on the physiological G protein coupling mechanism of EET receptors.
In conclusion, using the EET radiolabeled agonist 20-125I-14,15-EE8ZE, we identified a specific, monophasic, and reversible EET binding site on U937 cell membranes with high affinity for EETs and receptor density within a physiological range. The agonist binding was sensitive to guanine nucleotide modulation. These findings suggest that the 20-125I-14,15-EE8ZE binding site, which is most likely a GPCR, is a specific EET receptor.
Polymorphisms of sEH and P450s that result in decreased EET concentration have been correlated with pathogenesis of coronary artery disease, hypertension, and myocardial infarction (King et al., 2005; Liu et al., 2005; Lee et al., 2006). Thus, increasing EET activity is an exciting therapeutic strategy for the treatment of those diseases. Identification of an EET receptor(s)/binding site(s) will facilitate understanding fundamental EET receptor signaling mechanisms under physiological and pathological conditions and provide a useful tool to screen for EET agonists as potential therapeutic drugs.
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Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
ABBREVIATIONS: EET, epoxyeicosatrienoic acid; P450, cytochrome P450; BKCa, large conductance, calcium-activated potassium; 14,15-EE8ZE, 14(S),15(R)-cis-epoxy-eicosa-8Z-enoic acid; 14,15-EE5ZE,14,15-epoxyeicosa-5(Z)-enoic acid; 14,15-DHET, 14,15-dihydroxyeicosatrienoic acid; DHET, dihydroxyeicosatrienoic acid; sEH, soluble epoxide hydrolase; 20-I-14,15-EE8ZE, 20-iodo-14,15-epoxyeicosa-8(Z)-enoic acid; OTs, 20-tosyl; HPLC, high-performance liquid chromatography; TBSTM, Tris-buffered saline containing 0.1% Tween 20 and 5% milk; 15-HETE, 15-hydroxyeicosatetraenoic acid; Ro 20,1724, 4-[(3-butoxy-4-methoxyphenyl)-methyl]-2-imidazolidinone; AUDA, adamantyl dodecanoic acid urea; IBTX, iberiotoxin; GTPS, guanosine 5'-O-(3-thio)triphosphate; GPCR, G protein-coupled receptor; U46619
[GenBank]
, 9–11-dideoxy-11, 9a-epoxymethano-prostaglandin F2a.
The online version of this article (available at http://jpet.aspetjournals.org) contains supplemental material. 
Address correspondence to: Dr. William B. Campbell, Department of Pharmacology and Toxicology, Medical College of Wisconsin, 8701 Watertown Plank Rd., Milwaukee, WI 53226. E-mail: wbcamp{at}mcw.edu
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